A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine

Abstract

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

FIG 1

Characterization of the fusogenic and IS activities of FeLV Env and generation of a fusion-positive, immunosuppression-negative specific mutant. (A) Schematic representation of the FeLV Env. The left panel shows the three-dimensional structure of the Env trimer, with the surface (SU) and transmembrane (TM) subunits and the immunosuppressive domain (ISD). On the right, a more detailed representation shows the linear organization of the FeLV Env, with the furin cleavage site, the hydrophobic fusion peptide and the transmembrane anchor. The ISD amino acid sequence is shown for the MLV and FeLV Env proteins, with the E→R substitution indicated. (B) IS activities of FeLV and MLV Env wild-type and mutant proteins in the in vivo MCA205 tumor rejection assay. Tumor growth was monitored twice or thrice weekly and tumor areas (mm²) determined by measuring perpendicular tumor diameters. Results are expressed as described previously (35, 36) with an IS index calculated as (A_env − A_none)/A_none at days 7 and 8 postinjection, where A_env and A_none are the mean areas of tumors from BALB/c mice injected with cells expressing the FeLV or MLV env gene to be tested or with control cells transfected with an empty vector (none). (C) Infectivities of FeLV Env and its mutant derivative as expressed on the surface of MLV viral pseudotypes, using G355 cells as a target. Titers (LacZ-positive focus-forming units/ml of viral supernatant) were measured as described previously (38) (mean values ± standard deviations [SD]).

FIG 2

Vaccine-induced specific responses. (A) Vaccine-induced FeLV gag/pro/env (left)-, gag/pro (middle)- and env (right)-specific responses measured by IFN-γ ELISpot assay at 1 week after vaccination (day 35). The frequency of FeLV-specific IFN-γ-producing cells (expressed in log₁₀) was higher in vCP2296-vaccinated animals (KW test, P = 0.07 for env and P = 0.05 for gag/pro; ANOVA, P = 0.05 for env/gag/pro-specific responses). (B) Vaccine-induced FeLV gag/pro/env (left)-, gag/pro (middle)- and env (right)-specific responses measured by IL-10 ELISpot assay at 1 week after vaccination (day 35). The median frequency of FeLV Env-specific IL-10-producing cells (expressed in log₁₀) was higher in vCP2295-vaccinated cats (KW test, P = 0.02 for env-, P = 0.23 for gag/pro-, and P = 0.08 for env/gag/pro-specific responses). (C) Ratio of vaccine-induced FeLV gag/pro/env (left)-, gag/pro (middle)- and env (right)-specific IFN-γ-producing cells to vaccine-induced FeLV-specific IL-10-producing cells, expressed in log₁₀ (day 35). The median ratio was higher in vCP2296-vaccinated cats (KW test, P = 0.01 for env-, P = 0.04 for gag/pro, and P = 0.03 for env/gag/pro-specific responses). The box-and-whisker plots show the median, lower, and upper quartiles as well as extreme values.

FIG 3

FeLV proviral loads after challenge in the first (A) and the second (B) studies. Proviremia was expressed in logarithm (log) provirus copy number/50,000 WBCs. Green lines represent cats which were not persistently antigenemic. Red lines represent cats considered persistently antigenemic (cats found positive for circulating p27 on at least 5 occasions and/or positive at the end of the follow-up period).

FIG 4

Ratio of vaccine-induced FeLV-specific IFN-γ-producing cells to FeLV-specific IL-10-producing cells in cats protected against persistent antigenemia versus antigenemic cats, for the whole set of vaccinated animals (both vCP2295 and vCP2296). The data shown are for the env (A)-, gag/pro (B)-, and gag/pro/env (C)-specific responses at day 35. The median ratio was higher in cats that did not develop persistent antigenemia (KW test, P = 0.003 for env-, P = 0.02 for gag/pro-, and P = 0.004 for env/gag/pro-specific responses). The box-and-whisker plots show the median, lower, and upper quartiles as well as extreme values.